1. Field of the Invention
This invention relates to a method for determining antigens, antibodies and their complexes, qualitatively and quantitatively, and more particularly to a method for determining such substances which uses a chemiluminescent marker and an activator in a conventional heterogeneous assay.
2. Description of the Prior Art
The quantitative determination of antigens, antibodies and their complexes in secretions, excreta and body fluids of vertebrates and humans is of great importance in biology and medicine. These procedures can be of great utility in diagnosis, as well as for other applications.
It is known to detect an immunological reaction by marking one or more of the ligands with a radioactive isotope, or by conjugating it with an enzyme, a fluorescent dye or a chemiluminescent material such as luminol or luciferin.
Radioimmunoassay is disclosed in Journal of Clinical Endocrinology 27 (1967) p. 973 and Ibid. 28 (1968) p. 343. The essential disadvantage of this technique lies in the necessity of working with isotopes which emit radiation and in the necessarily expensive equipment needed to carry out such assays.
Marking a reaction component with an enzyme has the disadvantage that marking is complicated and the final reaction product is difficult to preserve and use. Furthermore, the enzymes used are biologically active substances of extremely complex nature, which is the basis for these difficulties. Finally, the substrates which are used in the ultimate determination of the bound enzymes are carcinogenic, which is evidently disadvantageous. The enzyme assay of this type is discussed in Bull. World Health Organ. 53 (1976).
In the fluorescence technique, the reaction products containing antigens and antibodies are detected by irradiation with light of short wavelength. In this case, the exciting light must be distinguished from the emitted light, which requires very expensive apparatus.
The chemiluminescent materials hitherto prepared for chemiluminescence investigation have been very difficult to bind to a ligand used in an immunological reaction, and, furthermore, up to 99.3% of the original luminescence is lost when the compounds are bound. This is disclosed in, e.g., Nature, Vol. 299 (1979), pp. 646-647. It is further reported in Journal of Immunological Methods 21, pp. 178-184, that the chemiluminescent material luminol is unsuitable for routine clinical laboratory tests for this reason.
The disclosures of U.S. Pat. No. 4,193,983 are to the contrary. In that patent, luminol is plainly disclosed to be a compound especially suited for this purpose. Hydrogen peroxide or calcium hypochlorite are mentioned as activators for chemiluminescence. Furthermore, in this U.S. Pat. No.the use of fluorescein isothiocyanate is disclosed for a homogeneous fluorescence assay.
In spite of intensive effort, it has hitherto been impossible to carry out a process of the type described above with satisfactory cost and efficiency. This is also true, for example, for the assay described in U.S. Pat. No. 4,238,195. In that patent, markers in the form of fluorescein and its derivatives were proposed, which require especially energy-rich materials for activation. These materials are prepared by a very complex reaction between derivatives of oxalic acid (oxalyl chlorides among others) and hydrogen peroxide, and their preparation requires a great expenditure of labor. Furthermore, it is not inconceivable that under certain conditions these materials might generate poisonous phosgene.
Hence, a need has continued to exist for a chemiluminescent marker for immunological reactions which avoids the drawbacks of the known materials.